Enzymatic degradation of organophosphates (OPs) is performed by specialized enzymes including bacterial organophosphorus hydrolase (OPH) and mammalian paraoxonase. Paraoxonase also referred to as, arylesterase (EC 3.1.1.2) is a 43 kDa molecular weight calcium dependent ester hydrolase that catalyses the hydrolysis of a broad range of esters such as OPs, and unsaturated aliphatic and aromatic carboxylic esters. Its name derives from the ability of this protein to hydrolyze paraoxon, the toxic metabolite of the insecticide parathion. In addition to paraoxon, paraoxonase is able to detoxify a number of other insecticides, e.g. diazonin, as well as the potent nerve gases sarin and soman that target acetylcholinesterase (AChE). The paraoxonase gene (PON) family consists of at least three members: PON1, PON2 and PON3, which are located on the human 7q21.3-22.1 chromosome. No significant endogenous expression of PON2 and PON3 genes has been detected. Most PON1 expression takes place in the human liver; from there the protein is secreted into blood where it circulates associated with high density lipoprotein (HDL) particles. Paraoxonase has the unusual property that the mature protein retains its hydrophobic N-terminal signal peptide, which is used as an anchor for association with HDL. The enzyme has three potential N-linked sites and carbohydrate accounts for approximately 16% of its molecular mass.
There is a significant variation in paraoxonase activity in the human population, which is a result of polymorphism in the PON1 promoter that leads to different levels of expression, as well as polymorphism in gene sequence that leads to allele forms of protein with different specific activity. Both types of polymorphisms are quite common among the human population generating a range of paraoxonase serum activity in the population. The apparent molecular mass of serum paraoxonase varies as the result of heterogeneous glycosylation.
Neither the function nor natural substrate(s) for paraoxonase have yet been identified. One possible substrate is oxidized low density lipoprotein (LDL) [1-3]. Paraoxonase has been shown both to prevent formation of oxidized LDL and to hydrolyze LDL-derived oxidized phospholipids. Since accumulation of oxidized LDL is one of the key factors in development of atherosclerosis, paraoxonase activity may correlate with development of this disease. For example, Shih et al demonstrated that PON1 −/− mice were extremely sensitive to diet-induced atherosclerosis in comparison with wild type mice. Since there is a significant variation in paraoxonase activity among the population, evaluation of paraoxonase levels of individuals may have a significant diagnostic value, predicting the chances, development and prognosis of atherosclerosis.
Another possible natural substrate is lipopolysaccharide (LPS) or mediators of septic shock. It has been shown that high density lipoprotein (HDL) can inactivate LPS [4]. Moreover, intraperitoneal injection of mice before and up to 2 hours after LPS administration afforded protection against septic shock [5]. In addition, PON-1 knockout mice are extremely sensitive to LPS [6].
Paraoxonase is able to hydrolyze a number of OP toxins in vitro, and the ability of paraoxonase to protect animals in acute OP poisoning has been extensively studied. Injection of purified paraoxonase protected animals against OP toxicity [7, 8]. Further proof of the ability of paraoxonase to protect animals has been obtained from studies on PON1 “knock-out” mice. Destruction of the PON1 gene by knock-out technology creates mice that lack paraoxonase. Compared to wild type littermates, PON1 deficient mice were extremely sensitive to the toxic effects of chlorpyrifos, an OP. Thus, monitoring of paraoxonase activity may help to evaluate a person's ability to withstand OP poisoning associated with deployment of chemical weapons.
Consequently, monitoring blood levels of paraoxonase may be used to identify a predisposition to atherosclerosis, sepsis and OP poisoning. However, the absence of a robust test for detection of paraoxonase levels in blood has significantly delayed progress in studying the diagnostic value of paraoxonase. There are two major options for detection of this enzyme activity. The first is a change in optical density and the second the generation of a fluorescent product.
Currently, the most common substrates for paraoxonase used in research are paraoxon and phenylacetate. Paraoxonase catalyzed hydrolyses of paraoxon leads to release of nitrophenol, which can be detected by monitoring adsorption at 405 nm. This reaction is used to measure paraoxonase activity in fundamental and clinical research. The main disadvantages of this substrate are the low Vmax of hydrolysis, which results in relatively low sensitivity and, due to its toxicity, paraoxon requires special handling conditions. The arylesterase activity of paraoxonase is usually measured through hydrolysis of phenylacetate. This reaction has a much higher Vmax, than the Vmax of paraoxon hydrolysis; however, phenylacetate is also hydrolyzed by a number of other esterases in cell extracts and serum samples, which significantly decreases the specificity of detection. In addition, the detection of phenylacetate hydrolysis is based on monitoring adsorption at 270 nm making paraoxonase detection difficult, or impossible, in protein rich solutions or in extracts containing detergents like Triton X-100.
The OPH gene was originally found in two soil microorganisms, Pseudomonas diminuta and Flavobacterium sp. It has been suggested that this enzyme evolved recently in these bacteria in response to industrial soil contamination with organophosphate compounds. Like paraoxonase, OPH catalyzes a broad range of organophosphate esters including sarin and VX. Due to this activity these organisms may have additional utility in decontamination of OPs in the environment. In this context a sensitive and robust assay would be necessary to confirm expression of OPH in the presence of a large excess of phosphatase activity. Thus, it is essential that the substrate has very little or no affinity for phosphatases.
The foregoing shows that there exists a need for detecting organophosphatase activity including paraoxonase with high specificity and sensitivity. There exists a need for substrates with high specificity for OPH and paraoxonase. The advantages of the present invention as well as inventive features will be apparent from the detailed description of the embodiments of the invention provided herein.